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Psoriasis is a multifactorial skin disease characterised by epidermal abnormalities and infiltration by lymphocytes and polymorphonuclear leukocytes (PMN). Skin-derived antileukoproteinase (SKALP), also known as elafin, is a potent inhibitor of human leukocyte elastase and proteinase 3, two PMN-derived proteinases implicated in tissue destruction and leukocyte migration. We have shown that, at least at the protein level, SKALP is significantly decreased in lesional skin of patients with pustular psoriasis compared with plaque-type psoriasis. This finding raised the possibility that SKALP could be one of the candidate genes for pustular forms of psoriasis. We therefore performed single strand conformation polymorphism (SSCP) analysis on the SKALP gene to screen for mutations/polymorphisms in the exons of 30 patients with plaque-type psoriasis, 15 patients with pustular psoriasis and 48 healthy controls. In exon 1 a polymorphism was detected at position +43 relative to the translation start site, resulting in a substitution of threonine for alanine in the signal peptide. In the promoter region a dinucleotide repeat polymorphism was identified. Both polymorphisms were not associated with pustular psoriasis, or psoriasis in general. Our data indicate that the decrease in SKALP activity in pustular psoriasis is not caused by mutations in the coding region of the gene, and that there is no allelic association between pustular psoriasis and SKALP gene polymorphisms.

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Keratinocytes of inflamed epidermis (psoriasis, wound healing) are hyperproliferative and display an abnormal differentiation programme. This regenerative differentiation pathway is characterized by the induction of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6, CK16, CK17, and the proteinase inhibitor SKALP/elafin. In the study reported here we investigated the induction and regulation of SKALP expression as a marker for regenerative differentiation in epidermal keratinocytes. Various cytokines and growth factors known to be present in psoriatic epidermis were examined for their ability to induce SKALP gene expression in cultured human keratinocytes. Tumour necrosis factor-alpha (TNF-alpha) and serum were found to be potent inducers of SKALP expression at both the mRNA and the protein levels. SB202190 or SB203580, two specific p38 MAP kinase inhibitors almost completely blocked the induction of SKALP expression by TNF-alpha and serum. These results suggest that in keratinocytes, p38 activity is crucial for the induction of SKALP gene expression. These findings could be relevant for the elucidation of the mechanisms involved in normal and disturbed epidermal differentiation.

Summary Impetigo herpetiformis (IH) is a rare pustular dermatosis with unknown aetiology, typically occurring during pregnancy. Based upon a similar clinical and histological presentation, i.e. spongiform accumulation of polymorphonuclear leucocytes in the stratum corneum, several authors consider IH as a variant of generalized pustular psoriasis (GPP), while others state that IH is a separate entity. Skin-derived antileucoproteinase (SKALP) is a strong and specific inhibitor of human leucocyte elastase (HLE) and proteinase 3, two neutral proteinases that have been implicated in leucocyte migration and tissue destruction. Previously, we reported decreased SKALP activity in pustular forms of psoriasis compared with plaque psoriasis. In this study we present a case study of a patient with IH, where SKALP activity was measured using biochemical and immunochemical techniques. Epidermal scales and sera were collected during the course of the disease. Comparison was made with three patients with GPP and six patients with plaque psoriasis. Initially, anti-HLE activity in epidermal scales of the patient with IH was comparable with values in patients with GPP, i.e. decreased compared with plaque psoriasis. During the course of the disease, anti-elastase activity dropped to undetectable levels, concomitant with the appearance of free elastase activity. This finding suggests a total saturation of epidermal anti-HLE activity. Low levels of SKALP, presumably complexed with HLE, could be measured immunochemically in scale extracts. Serum levels of total SKALP correlated with the disease activity. We suggest that a reduced amount of epidermal SKALP contributes to an imbalance between elastase and its inhibitor, resulting in the formation of epidermal pustules. This mechanism of pustule formation could apply both to GPP and IH, suggesting a final common pathway in the pathogenic mechanisms of IH and GPP.

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Item does not contain fulltext Katholieke Universiteit Nijmegen, 13 juni 2000 Promotor : Kerkhof, P.C.M. van de Co-promotor : Schalkwijk, J. 214 p.

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Recently, several new genes have been discovered in various species which are homologous to the well-characterized human epithelial proteinase inhibitor elafin/SKALP (skin-derived anti-leukoproteinase). Because of the high degree of conservation and the similarities in genomic organization, we propose that these genes belong to a novel gene family. At the protein level, the family members are defined by: (1) an N-terminal domain consisting of a variable number of repeats with the consensus sequence Gly-Gln-Asp-Pro-Val-Lys that can act as an anchoring motif by transglutaminase cross-linking, and (2) a C-terminal four-disulphide core or whey acidic protein (WAP) domain, which harbours a functional motif involved in binding of proteinases and possibly other proteins. We have proposed the name trappin gene family as a unifying nomenclature for this group of proteins (trappin is an acronym for TRansglutaminase substrate and wAP domain containing ProteIN, and refers to its functional property of ‘getting trapped’ in tissues by covalent cross-linking). Analysis of the trappin family members shows extensive diversification in bovidae and suidae, whereas the number of primate trappins is probably limited. Recent biochemical and cell biological data on the human trappin family member elafin/SKALP suggest that this molecule is induced in epidermis by cellular stress. We hypothesize that trappins play an important role in the regulation of inflammation and in protection against tissue damage in stratified epithelia.

Secretory leukocyte protease inhibitor (SLPI) is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte-derived proteinases, has anti-HIV-1, antibacterial, and anti-fungal properties, and interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages. We now report that at both the mRNA and the protein level, SLPI shows inducible expression in a nonglandular epithelium. A weak expression of SLPI was found in the stratum granulosum of adult normal human epidermis; however, in lesional psoriatic epidermis and in migrating keratinocytes of healing wounds, a strong cytoplasmic staining was seen in the suprabasal keratinocytes. Remarkably, in the dermis adjacent to SLPI-expressing keratinocytes, SLPI was found extracellularly associated with elastin fibers, whereas the dermis in normal skin was negative. In cell culture, SLPI was hardly expressed in monolayers of proliferating keratinocytes. Differentiating cultures with a phenotype of normal skin expressed low levels of SLPI, whereas cultures with a regenerative/psoriatic phenotype expressed high levels. Functional studies with recombinant SLPI indicated that its anti-bacterial spectrum and potency are distinct from other anti-microbial peptides such as lysozyme and defensins. In view of the multiple functions of SLPI and the inducibility, we propose that it acts as an important first line defence mechanism in cutaneous injury.

In this study, the immunohistochemical expression of a new inducible elastase inhibitor, SKALP (skin-derived anti-leucoproteinase)/elafin, in the tissue of squamous cell carcinoma and uninvolved oesophageal mucosa was studied using a polyclonal rabbit anti-serum against SKALP/elafin. The results were compared with the immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and the TUNEL assay in serial sections. In non-malignant oesophageal mucosa, the expression of SKALP/elafin was localized in the cells of the stratified zone overlying the PCNA-positive basal zone. In oesophageal cancer, the incidence of the expression was significantly related to the degree of the differentiation of the tumour. Characteristically, the expression was almost limited in tumour cell nests that had a clear squamous phenotype. In tumour cell nests, the expression of SKALP/elafin was localized in the cells overlying PCNA-expressing cells and no expression was found in the cells that expressed PCNA; DNA fragmentation was often observed in the same cell layers as those in which SKALP/elafin immunoreactivity was found. This enzyme inhibitor is speculated to be involved in the induction of the cell differentiation and apoptosis of human squamous cell carcinoma cells of the oesophagus. Images Figure 1 Figure 2 Figure 3 Figure 4

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Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

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Contains fulltext : mmubn000001_211738271.pdf (Publisher’s version ) (Open Access) Promotores : P. van de Kerkhof, B. Wieringa en J. Schalkwijk 131 p.

The epidermal serine proteinase inhibitor SKALP (also known as elafin), directed against human leukocyte elastase and proteinase 3, is strongly induced in suprabasal keratinocytes during inflammation, The presence of SKALP/elafin in urine has been demonstrated for several inflammatory skin disorders, such as psoriasis, erythroderma, and erysipelas. In this study we investigated whether SKALP/elafin levels in serum and urine of psoriatic patients can be used as a marker for disease activity during treatment. Patients with severe chronic disabling psoriasis were treated for 16 weeks with cyclosporin A, which resulted in a marked clinical improvement as measured with the PASI score. SKALP/elafin levels both in serum and urine were determined with an enzyme-linked immunosorbent assay (ELISA). Measurements were performed at the start of the cyclosporin A treatment, and after regular intervals up to 16 weeks, The results indicate that 1) SKALP/elafin determination in serum rather than in urine is the preferred method, because the decrease in serum SKALP levels during therapy is more pronounced and correlated better with the clinical course of the patients; 2) SKALP/elafin levels in serum decreased during cyclosporin A treatment (p < 0.05); and 3) SKALP/elafin levels in serum correlate with the PASI score (p < 0.01). We conclude that SKALP/elafin measurement in serum of patients with severe psoriasis provides a tool for monitoring disease activity.